Chapter 10 - Genetic Engineering: A Revolution in Molecular Biology

Mycobacterium tuberculosis

altered by transduction with genes for luciferase, an enzyme from jellyfish that causes light emission; used to assay drug resistance (live cells light up)

RNAi Example

block a gene that controls tomato ripening - develops better flavor, ripened with ethylene; developing canola (rapeseed) oil plant with more saturated fat, so that margarine can be made w/o artificial hydrogenation; tobacco plants that synthesize human antibodies

PQ: Restriction endonucleases are enzymes which cut DNA at a specific palindromic sequence. They are produced _______________________.

by bacteria to protect against viral infection

PQ: The two strands that compose the DNA double helix can be separated by _________________________.

denaturation by raising the temperature

PQ: Gel electrophoresis separates DNA fragments using __________.

electric current

PQ: In order for biotechnology methods to produce human proteins from bacterial cells in culture, all the following must occur

mRNA must be isolated, a cDNA copy must be made, and polymerase makes the DNA double-stranded

Gene Probes

made from oligonucleotide tracers detect specific nucleotide sequences in unknown samples

Restriction endonucleases non-menclature

named by combining the first letter of the genus + first two letter of the sp. + the endonuclease #. Examples: E. coli, the 1st discovered; Haemophilus influenzae type d, the 3rd

pq: Southern blotting finds specific DNA fragments _____________.

on a filter

Transfection

process of introducing foreign genes into organisms

Transgenic

recombinant organisms produced this way

PCR method: Thermal Recycler

regulates cyclic temperature changes

PQ: DNA fragments separate on an electrophoresis gel with the ___________________.

smallest segments traveling furthest

Two different nucleic acids can hybridize, what are they?

ssDNA and ssRNA

PQ: When a restriction enzyme makes a staggered cut across a strand of DNA, this is known as a ________________.

sticky end

PQ: The most detailed analysis of DNA comes from determining ______________.

the actual order and types of bases in DNA

PQ: A quick way to determine whether bacterial colonies have incorporated a gene of interest is _____________.

to do a hybridization test

FISH - fluorescent in situ hybridization

uses probes directly applied to cells and observed microscopically to detect specific genetic markers used to identify bacteria

Pseudomonas syringae

wild strain promotes ice or frost formation on moist plant surfaces; alteration using recombinant plasmids created a different strain that could prevent ice crystals from forming by outcompeting pathogenic P. syringae: Frostban - used to stop frost damage in strawberry and potato crops

Transgenic Plants

"Agrobacterium tumefaciens" - a natural tumor-producing bacterium; Ti plasmid inserts into the genomes of the infected plant cells

Vector in Recombinant DNA Technology

-Plasmid: small, well-characterized; easy to manipulated; transferred to the host by transformation; bacteria: BACs, E. coli; yeast: YACs; Saccharomyces cerevisiae plasmids (YACs) - able to copy much larger stretches of foreign DNA -Virus: retroviruses, adenoviruses and herpes viruses used in gene therapy -Bacteriophages: cess of transduction; cosmid - plasmid within a phage

Explain the process of "Construction of a Recombinant, Insertion, and Genetic Expression"

-Prepare the isolated genes for splicing into a vector by digesting the gene and the plasmid with the same restriction endonuclease enzymes creating complementary sticky ends on both the vector and insert DNA -The gene and plasmid are placed together, their free ends base-pair and ligase joins them -The gene and plasmid combination is a recombination -The recombinant is introduced into a cloning host -The cloning host transcribes the gene and translates the protein (purifies protein)

Explain the process of PCR

-Primers of known sequence are added, to indicate where amplification will begin, along with special heat tolerant DNA polymerase and nucleotides -Repetitively cycled through denaturation, priming, and extension -Each subsequent cycle doubles the number of copies for analysis -Essentially important in gene mapping, the study of genetic defects and cancer, forensics, taxonomy, and evolutionary studies

RNAi

-RNA interference: inserting a strand of antisense RNA -Process of turning off each gene tells us what that codon/protein did to the organisms

Desirable Features in a Cloning Host

-Rapid turnover, fast growth rate -Can be grown in large quantities using ordinary culture methods -Nonpathogenic -Genome that is well delineated (mapped) -Capable of accepting plasmid or bacteriophage vectors -Maintains foreign genes through multiple generations -Will secrete a high yield of proteins from expressed foreign genes

Plasmids

-Small -Well characterized -Easy to manipulate -Can be transferred into appropriate host cells through transformation

Sanger technique for sequencing DNA

-Test strands are denatured to serve as a template to synthesize complementary strands -Fragments are divided into tubes that contain primers, DNA polymerase, all 4 nucleotides, and fluorescent labeled dideoxynucleotide

Recombinant DNA Technology

-The intentional removal of genetic material from one organism and combining it with that of a different organism -Objective: cloning which requires the desired donor gene be selected, excised by restriction endonucleases, and isolated -The gene is inserted into a vector (plasmid, virus) that will insert the DNA into a cloning host -Cloning host is usually bacterium or yeast that can replicate the gene and translate it into a protein product

Transgenic Animals

-Use a virus to transfect a fertilized egg or early embryo *"Mouse Model" to express everything like CF, Alzheimer's, sickle cell anemia -Transgenic animals will transcribe and translate eukaryotic genes *Sheep/goats are easier to work with because they are bigger, have more blood and more proteins

Southern Blot

1. DNA fragments = RFLP separated by electrophoresis 2. Fragments are made visible, and a nylon filter is placed on the gel that binds the DNA 3. Radioactive DNA probes are added; wherever it hybridizes it will bind 4. X-ray film used to visualize the hybridization

Thermal Recycler steps

1. DNA sample - small DNA fragment 2. Denaturation - separation into 2 strands with heat to 94 C 3. Priming - system is cooled to 60 C and primers - synthetic oligonucleotides of known 15 -30 bps - added that will attach at ends of strands to promote replication of amplicons 4.Extension - 72 C; DNA polymerase isolated from thermophilic bacteria

PQ: Human beings contain approximately ______ genes.

20,000-25,000

How many different Restriction endonucleases have been discovered?

>3000 in bacteria; >600 available commercially and used routinely

PQ: DNA fingerprinting was first devised by _______________.

Alex Jeffries

What is one the problems with the Thermal Recycler method?

Amplification of non-target areas through contamination demand a clean environment & specific primers and special enzymes that degrade the contaminating DNA before it is amplified

PQ: The polymerase chain reaction is a method of ________________.

DNA amplification

PQ: The PCR technique operates by cycling through three basic steps. These three steps include:

Denaturation, priming, and extension

Restriction fragment length polymorphisms (RFLPs)

Differences in the cutting patterns of specific restriction endonucleases; Allows us to compare DNA of different organisms at specific sites

PQ: Which of the following is NOT a type of cloning vector used to transfer recombinant DNA into a cloning host? A) plasmids B) bacteriophages C) cosmids D) E. coli

E. coli

Restriction endonucleases

Enzymes capable of recognizing foreign DNA and breaking the phosphodiester bonds between adjacent nucleotides on both strands of DNA; breaks the DNA apart

PQ: Bioengineered hormones, enzymes, and vaccines are easier to produce but not as safe as their natural equivalents. True or False?

False

PQ: Genetic engineering is also called bioengineering or biotechnology. True or False?

False

PQ: Scientists are working to find a cure for cancer. This is an example of basic science. True or False?

False

True or False: Produced by bacteria and protozoa to protect against viral infection

False - only bacteria

PQ: A cloning host has the following desirable features _________________________.

Fast growth rate., well-mapped genome, and keeps foreign genes intact.

PQ: From the one strand noted, identify the palindromic sequence below. A) GAATTC B) GCAT C) TTAGAA D) TAGAT

GAATTC

PQ: Correction or repair of faulty genes in humans is termed ______________.

Gene Therapy

PQ: The systematic study of an organism's genes and their functions is called

Genomics

Bacteriophages

Have the natural ability to inject their DNA into bacterial hosts through transduction

PQ: Transgenic animals can be engineered to produce ___________.

Human Protein

PQ: Gene therapy is said to be _____ if cells are altered within the body.

In vivo

Complementary DNA (cDNA)

Made from any type of RNA (mRNA, tRNA, rRNA, etc.); Used to synthesize eukaryotic genes from mRNA transcripts and is free from introns

"Sticky Ends"

Make staggered symmetrical cuts that leave short tails called ___________. Cut four to five bases on the 3' strand and on the 5' strand, leaving overhangs on each end. Can accept complementary tails for gene splicing. Adhesive tails will base-pair with complementary tails on other DNA fragments or plasmids.

Polymerase Chain Reaction (PCR)

Method to amplify DNA; rapidly increases the amount of DNA in a sample

Ligase

Necessary to seal the sticky ends together; Used in the final splicing of genes into plasmids and chromosomes

PQ: During recombinant DNA technology DNA is inserted by a vector. Identify a vector.

Plasmids

What does PCR stand for?

Polymerase Chain Reaction

PQ: In hybridization, oligonucleotides function as ____________.

Probes

Gel Electrophoresis purpose

Produces a readable pattern from DNA fragments

PQ: Typically, the final product of recombinant DNA technology is a ____________.

Protein

Gel Electrophoresis testing

Samples are placed in compartments in a soft agar gel and subjected to an electrical current; Phosphate groups have a negative charge, which causes DNA to move toward the positive pole in the gel; Larger fragments migrate more slowly; smaller fragments migrate more quickly; Position of fragments are determined by staining the gel; Creates a genetic fingerprint

What happens with probes hybridize with an unknown area of DNA or RNA?

They tag the precise area and the degree of hybridization helps determine the nature of the nucleic acid

PQ: The vector is inserted into a cloning host by __________.

Transformation

PQ: Palindromes are sequences of DNA that are identical when read from the 5' to 3' direction on one strand of DNA and from the 5' to 3' direction on the other strand. True or False?

True

PQ: The primary intent of recombinant DNA technology is to deliberately remove genetic material from one organism and combine it with that of a different organism. True or False?

True

PQ: Transgenic organisms are produced through recombinant DNA technology and can be patented. True or False?

True

Reverse Transcriptase

Used to convert RNA into DNA; Copies called complimentary cDNA (cDNA); Has a role in the replication of the AIDS virus

Cloning Host

Usually bacterium or yeast that can replicate the gene and translate it into a protein product (Ex. E. Coli, Saccharomyces cerevisiae, or animal cell culture & even live animals & plants)

Name a few Biochemical Products that have been made from Recombinant DNA Technology.

-Enables large scale manufacturing of life-saving hormones, enzymes and/or vaccines *Insulin for diabetes *Human growth hormone for dwarfism *Erythropoietin for anemia *Factor VIII for hemophilia (blood doesn't clot naturally) *HBV vaccine

Nucleic Acid Hybridization and Probes

-Method for analysis of DNA -Single stranded DNA can unite with other single-stranded DNA or RNA, and RNA can unite with other RNA - hybridization -Foundation for gene probes - short DNA fragments of a known sequence that will base-pair with a stretch of DNA with a complementary sequence, if one exists in the sample -Useful in detecting specific nucleotide sequences in unknown samples

Vector considerations

-Origin of replication is needed so it will be replicated -Vector must accept DNA of the desired size -Gene which confers drug resistance to their cloning host